Activation of human CD8+ T-cells with nitroso dapsone-modified HLA-B*13:01-binding peptides

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T-cells. However, the nature of the antigenic determinants that interact with HLA, and whether T-cell stimulatory peptides contain the bound drug metabolite has not been defined. Since susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T-cells from hypersensitive human patients. Cysteine-containing 9mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL[Pep2] and AQDAEACAL [Pep3]) and the cysteine residue was modified with nitroso dapsone. CD8+ T-cell clones were generated and characterized in terms of phenotype, function and cross-reactivity. Autologous antigen presenting cells and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. HPLC/LCMS confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. Antigen presenting cell HLA-B*13:01-restricted nitroso dapsone-modified Pep1 (n=124) and Pep3 (n=48) responsive CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified-Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterise a drug metabolite hapten CD8+ T-cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.


Introduction
Dapsone is a widely used antibacterial agent and is frequently prescribed for the treatment of leprosy. However, exposure to dapsone is associated with the development of a hypersensitivity syndrome, characterised by fever, skin rash, hepatitis, and generalized lymphadenopathy, in 0.5-3.6% of treated patients (1,2). Recent studies have shown that expression of human leukocyte antigen (HLA) 2 -B*13:01 is associated with increased risk of dapsone hypersensitivity (3)(4)(5)(6).
Dapsone contains aromatic amine groups that are susceptible to acetylation by Nacetyltransferase enzymes, a process of detoxification that limits the lifespan of the drug. The aromatic amine groups also undergo cytochrome P450-mediated hydroxylation yielding dapsone hydroxylamine (7). Nitroso dapsone is generated via auto-oxidation of the hydroxylamine metabolite. Nitroso dapsone is protein-reactive and covalently modifies cysteine residues on proteins (8)(9)(10)(11).
Peripheral blood mononuclear cells (PBMC) from hypersensitive patients expressing HLA-B*13:01 are stimulated to proliferate with both dapsone and its nitroso metabolites (12,13). Through the generation of clones, HLA class II-restricted CD4+ and HLA-class I-and II-restricted CD8+ T-cells were shown to be stimulated with dapsone and nitroso dapsone via different pathways (13,14). The T-cell response to parent drug and metabolite was polyclonal with T-cells displaying a variety of TCR sequences (14). Dapsone interacts directly with HLA to trigger T-cell receptors, whereas nitroso dapsone triggers T-cell receptors though a pathway dependent on antigen processing, presumably through the formation of protein adducts. Both dapsone and nitroso dapsone interact with multiple HLA class I proteins to activate CD8+ T-cells, which makes it challenging to focus research on the drug HLA-B*13:01 interaction and specifically determine whether drug metabolite-modified peptides activate T-cells in an HLA allele-restricted manner. Therefore, the aim of this work was to (1) design HLA-B*13:01 binding peptides that contain a reactive cysteine residue (2), generate nitroso dapsone-modified peptides that are free of dapsone and the nitroso metabolite, and (3) explore immunogenicity of the peptides using autologous antigen presenting cells (APC) and APC transfected with the single HLA-B allele HLA-B*13:01.

Synthesis of drug modified peptides
Three 9mer peptides were identified as potential HLA-B*13:01 high affinity binders using the MHC binding prediction tool obtainable at www.iedb.org (15). Peptides were designed to each contain a single cysteine residue at sites distal from the HLA-B*13:01 binding motifs (glutamine at P2 and leucine at P9), as previous studies have shown that nitroso dapsone binds covalently to cysteine (11). Poly-alanine was chosen as peptide backbone to minimize the interaction between peptides and TCR. Cysteine was inserted within the binding motif to generate positional derivatives. To improve peptide solubility, glutamic acid and aspartic acid were also added. Aspartic acid is also a secondary anchor in position 3. All three peptides showed favourable binding to HLA-B*13:01 (a percentile rank of < 1 as predicted by NetMHC, Immune Epitope Database). The strategy for peptide design and predicted HLA-B*13:01 binding affinity is summarized in Figure 1A and B. Peptides were synthesized with an Fmoc protecting group at the N-terminus to avoid a non-specific reaction between nitroso dapsone and the primary amine of the peptide. The protecting group was removed before any functional analyses. Characterisation of the peptides was performed by mass spectrometry (16).
Mass spectrometric characterisation of dapsone nitroso modified peptides-Nitroso dapsone-modified peptides were characterized using a TripleTOF 6600 (AB Sciex) mass spectrometer. Briefly, samples were delivered into the mass spectrometer by automated in-line reversed phase liquid chromatography, using an Eksigent NanoLC 400 System (AB Sciex) mounted with a trap and analytical column (15 cm X 75 μm). A NanoSpray III source was fitted with a 10 μm inner diameter PicoTip emitter (New Objective). A gradient of 2-50% (v/v) acetonitrile/0.1% (v/v) formic acid over 90 min was applied to the column at a flow rate of 300 nl/min. Spectra were acquired automatically in positive ion mode using information dependent acquisition, using mass ranges of 400-1600 Da in MS and 100-1400 Da in MS/MS. Up to 25 MS/MS spectra were acquired per cycle (approximately 10 Hz) using a threshold of 100 counts per s, with dynamic exclusion for 12 s and rolling collision energy.
Quantification of drug molecules remaining in the peptide fractions-Free dapsone/dapsone nitroso remaining in the drug-modified peptide fractions were quantified by mass spectrometry. Calibration standards were prepared at the following concentrations (5-500 nM). All samples were diluted in 0.1% formic acid prior to analysis and spiked with the internal standard sulfamethoxazole (250 nM). Samples and standards were analysed immediately by a QTRAP5500 mass spectrometer (AB Sciex) coupled with an Ultimate 3000 LC system (Dionex Corporation, Sunnyvale, California). The multiple reaction monitoring transitions for each analyte were as following: dapsone 249.1/156.1, dapsone nitro 263.1/156.1, and the internal standard 254.1/156.1. Other mass spectrometry (MS) parameters, such as voltage potential and collision energy were optimized to achieve great sensitivity. Data acquisition and quantification were performed using Analyst 1.5 software.

Human subjects
Venous blood samples (50ml) were taken from 2 dapsone hypersensitive patients with positive (i) dapsone patch test and (ii) dapsone and nitroso dapsone lymphocyte transformation test as reported previously (14). Patient 8 (female, 28 years old at time of adverse event) displayed fever and abnormal liver function tests following 17-day exposure to dapsone. Patient 14 (male, 24 years old at time of adverse event) displayed fever, skin rash (erythema) and abnormal liver function tests following 21-day exposure to dapsone. Both individuals expressed HLA-B*13:01; the full patient HLA profiles are available in Zhao et al (14). Approval for the study was acquired from Shandong Provincial Institute of Dermatology and Venereology and informed written consent was obtained. A material transfer agreement was signed prior to transport of PBMC to Liverpool.
Anti-dapsone antibody production-Ovalbumin-dapsone conjugates were prepared by the reaction of nitroso dapsone with ovalbumin at a molar ratio of 10:1 (drug to protein) using methods as previously described (17). Antibody production was performed by Kaneka Eurogentec S. A. (Belgium) using a speedy 28-polyclonal package. Detailed information is available online (eurogentec.com).

Generation of nitroso-dapsone-modified peptide-responsive T-cell clones
PBMC (1 x 10 6 /well) were cultured in a 48-well plate with nitroso dapsone-modified Pep1 (patient 8 and 14) and Pep3 (patient 14 only due to limitations on the availability of the modified peptide) for 14 days to enrich the number of responsive T-cells prior to serial dilution. Peptide concentrations (10-50 μM) were selected based on a lack of intrinsic toxicity and no inhibition of phytohaemagglutinin (PHA)-treated healthy donor PBMC proliferation. Pep2 was reserved for crossreactivity studies as it was synthesized in low yield. T-cell clones were generated by serial dilution and repetitive mitogen-driven expansion. Briefly, irradiated allogenic PBMC (5 × 10 4 cells/well) in medium containing PHA and IL-2 were added to 96 well U bottom plates. T-cells were then diluted and added to the PBMC mixture at 0.3, 1 and 3 cell/well. Cultures were incubated for 14 days (37°C / 5% CO 2 ) and medium was supplemented with IL-2 every 2 days. On day 14, the growing clones were restimulated with PHA and irradiated allogenic PBMC (5 × 10 4 cells/well) in IL-2 containing medium and expanded for a further 14 days prior to testing for peptide specificity.

Antigen presenting cells
PBMC from the hypersensitive patients were cultured with supernatant from EBV-producing B-958 cells in the presence cyclosporine-A to generate immortalized autologous B-cell lines using established methods (14).
To generate a HMy2.C1R-HLA-B*13:01-P2A-B2M cell line, the pLJM1-EGFP plasmid (gift from David Sabatini Addgene plasmid # 19319) was modified by replacing the EGFP with a Multiple Cloning Site, adding an Eμ enhancer (pLJM1-Eμ-SFFV-NewMCS) and a P2A sequence. B2M was PCR amplified from cDNA from a volunteer and ligated into the distal end of the P2A sequence using restriction sites Age1/SnaB1. HLA-B*13:01 was PCR amplified from the HLA-B*13:01 pcDNA3.1(+) plasmid and ligated into the proximal end of the P2A sequence using restriction sites PME1 and EcoR1 to create the pJLM1-HLA-B*13:01-P2A-B2M plasmid. The pJLM1-HLA-B*13:01-P2A-B2M plasmid was transfected into competent E. coli and colonies grown on agarose plates under carbenicillin selection overnight at 37°C. Positive colonies were identified by colony PCR and grown up overnight in a shaking incubator at 37°C. Plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen). Correct integration was confirmed by Sagner sequencing. The HEK293 cell line was used as the machinery to generate 2nd generation lentivirus. HLA-B*13:01-P2A-B2M lentivirus was collected from the supernatant 96 h post transfection. HMy2.C1R cells were transduced with the HLA-B*13:01-P2A-B2M lentivirus supernatant and selected for positive transduction with puromycin. HMy2.C1R cells (ECACC 94050320), an EBV transformed B-cell line lacking HLA-B expression, was purchased from the UK Health Security Agency culture collections (https://www.culturecollections.org.uk).

Specificity testing of T-cell clones
Expanded T-cell clones (5 × 10 4 cells/well) were incubated with irradiated autologous antigen presenting cells (EBV-transformed B-cells; 1 × 10 4 cells/well) and modified peptides (10 μM) for 48 h in duplicate cultures, and proliferation was measured through addition of [ 3 H]-thymidine (0.5 μCi/well) for the final 16 h of the culture period. Peptide-free medium was used as negative control.

Characterization of drug-modified peptide-responsive T cell clones
T cell clones were phenotyped using flow cytometry for the CD4+ (CD4-APC (clone RPA T4)) and CD8+ surface receptors (CD8-PE (clone HIT8a)). T-cell clones (5 × 10 4 cells/well) were tested in dose-response studies for cross-reactivity with irradiated antigen presenting cells (1 × 10 4 cells/well) and nitroso dapsone-modified peptides (Pep1, 2 and 3; 1 to 100 μM) in triplicate cultures. Unmodified peptides subjected to the same culture conditions and purification steps were used as a negative control (1 to 100 μM). Clones were also cultured with soluble nitroso dapsone (1-40 μM; higher concentrations induced toxicity). Proliferation of the clones was measured by addition of [ 3 H]-thymidine for the final 16 hrs of the experiment.
The secretion of IFN-γ from the clones was assessed using ELIspot. T-cell clones were incubated with antigen presenting cells in the presence and absence of nitroso dapsonemodified peptides and other study compounds in IFN-γ antibody-coated ELIspot plates (37°C, 5% CO 2 ) for 48 hrs. Plates were then developed according to the manufact rer's instructions (Mabtech, Stockholm) and spots were counted using an AID ELIspot reader. To confirm levels of cytokine produced, the supernatant of 4 clones (2 drugspecific, 2 not drug specific) was analysed using a cytokine bead array according to the manufacturer's instructions (LEGENDplex, Biolegend Custom Human 11-plex panel). Briefly, supernatant pooled from triplicate wells for each clone was added to specific antibody-coated beads forming an analyte-antibody complex. After washing, a biotinylated detection antibody cocktail was added which bound to the specific analyteantibody complexes. Streptavidin-phycoerythrin was subsequently added which bound to the biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the bound analyte amount. Fluorescent signals were measured using BD FACSCanto II and analysed using LEGENDplex data analysis software where concentrations of each analyte are determined using a standard curve generated in the same assay.
In order to determine whether the detected activation of clones with nitroso dapsonemodified peptides was due to residual dapsone or nitroso dapsone or degradation of the peptides and liberation of free dapsone, clones (5 × 10 4 cells/well) were cultured with irradiated antigen presenting cells (1 × 10 4 cells/well) (i) in the presence of soluble dapsone (125-500 μM) and proliferation was measured by addition of [ 3 H]thymidine; and (ii) in the presence of nitroso dapsone-modified Pep1 or soluble nitroso dapsone and glutathione (1 mM), which binds covalently to the nitroso metabolite preventing protein binding (11), and proliferation was measured by addition of [ 3 H]thymidine. The irreversible binding of nitroso dapsone to antigen presenting cells was measured in the presence and absence of glutathione (1 mM) using immunofluorescence staining with an anti-dapsone antibody.
To explore the importance of HLA proteins in T-cell activation, T-cell clones (5 × 10 4 cells/ well) were cultured with Pep1 or Pep3 (i) in the absence of antigen presenting cells; (ii) in the presence of C1R-B*13:01 or C1R-parental antigen presenting cells (1 × 10 4 cells/well); and (iii) in the presence of C1R-B*13:01 antigen presenting cells pre-treated with either isotype (IgG1) or HLA class-I (DX17) or HLA class-II (Tu39) blocking antibodies for 30 min. T-cell proliferation or IFN-γ release were measured using [ 3 H]thymidine or ELIspot, respectively.

Synthesis and analysis of nitroso dapsone-modified peptides
HLA-B*13:01 designer peptides were created by incorporating HLA-B*13:01 anchor residues and a cysteine residue ( Figure 1A). These peptides are predicted as strong binders using the MHC binding prediction tool (pep1 0.862 μM; pep2 1.155 μM; pep3 1.175 μM; Figure 1 B). Nitroso dapsone-modified peptides were prepared from conjugation of Nterminal Fmoc protected peptides with the nitroso metabolite. This procedure was followed by Fmoc deprotection and HPLC purification. The final products were essentially free of soluble dapsone, nitroso dapsone or unmodified peptide based on HPLC analysis ( Figure  2A Figure 2D. To explore how the nitroso dapsone-modification affects the binding affinities of the peptides two approaches were adopted. First, the cysteine residue of all three peptides were replaced with bulky aromatic amino acids (phenylalanine [F] and tryptophan [W]) to mimic the nitroso dapsone modification in the cysteine containing peptide and B*13:01 binding affinity was assessed using NetMHCpan BA 4.1. It was not possible to assess drug peptide modifications directly. Second, in silico models of C and W containing peptides binding to HLA-B*13:01 were generated and compared to the predicted binding of the equivalent nitroso dapsone-modified peptide. Interestingly, when the cysteine residues of the designer peptides were replaced with the bulky aromatic amino acids, the binding of these peptides to HLA-B*13:01 was stronger than the cysteine containing peptides ( Figure 3A), suggesting that nitroso dapsone-modified peptides would be good binders to HLA-B*13:01. Molecular docking of AQDCEAAAL, AQDWEAAAL, and AQDC(DDS)EAAAL to a homology model of HLA-B*13:01 demonstrated that all these peptides could bind to HLA-B*13:01 similarly within the binding groove ( Figure 3B). Of particular interest, the predicted conformation of dapsone modified peptide AQDC(DDS)EAAAL is similar to the peptide AQDWEAAAL ( Figures 3C and D), with the bulky aromatic groups pointing out the binding groove to ensue favourable interaction of P2 and P9 anchor residues with HLA-B*13:01.

Generation and phenotypic assessment of nitroso dapsone-modified Pep1 and Pep3 T-cell clones
Initial testing of almost 400 T-cell clones derived from nitroso dapsone-modified Pep1 or Pep3-treated PBMC involved culture of T-cells with antigen presenting cells and the peptides or medium (as a negative control) in duplicate culture and comparison of proliferation. Almost 50% of the clones generated displayed reactivity against either nitroso dapsone-modified Pep1 (n=124) or Pep3 (n=48) and the strength of the proliferative response varied from a stimulation index (proliferation in test incubations with antigen / proliferation in control incubations with medium) of 2 to above 30 ( Figure 4). These T-cell clones were expanded and analysed for CD phenotype. Clones expressing the CD8+ receptor, with no CD4 expression were used in the experiments described below.

Functional characteristics of nitroso dapsone-modified Pep1-and Pep3-responsive CD8+ T-cell clones
A panel of up to 30 T-cell clones from both patients were utilized to assess cross-reactivity. All clones were stimulated to proliferate with nitroso dapsone-modified Pep1 or Pep3 with no discernible difference between the strength of the induced response observed ( Figure  5A); however, proliferative responses were not detected when the clones were cultured with unmodified peptides ( Figure 5B; shows Pep1 data). All clones were also activated with soluble nitroso dapsone, which forms adducts with protein in the cell culture assay ( Figure  7B), and the strength of the maximal response induced was similar with nitroso dapsone and nitroso dapsone-modified peptides ( Figure 5C).
Three clones were used in an ELIspot assay to study IFN-γ with Pep2. Clones secreted IFN-γ in the presence of all 3 nitroso dapsone-modified peptides (Pep1, Pep2 and Pep3; Figure 6A; the number of experiments with Pep2 was limited as the peptide was synthesized in small quantities). To confirm levels of cytokine produced, the supernatant from 4 clones (2 drug-specific, 2 not drug specific) was analysed using a cytokine bead array. Clones that were stimulated to proliferate also secreted IFN-γ, IL-5, Il-13, perforin and granzyme B (figure 6B-E).
Given the complete cross-reactivity profile of the clones with different peptides, well growing clones from either patient were selected and tested with Pep1 or Pep3 for the HLA restriction studies detailed below.

Nitroso dapsone-modified peptide-responsive CD8+ T-cell clones are not activated with residual dapsone or nitroso dapsone
To confirm that T-cell activation with the nitroso dapsone-modified peptides was not due to residual dapsone, clones were cultured with an optimal concentration of the parent drug. Clones were stimulated to proliferate with nitroso dapsone-modified peptide and soluble nitroso dapsone; however, proliferative responses were not detected with dapsone itself ( Figure 7A). Glutathione was used to differentiate between nitroso dapsonemodified peptide and soluble nitroso dapsone T-cell proliferative responses. The addition of glutathione to cell culture medium inhibits the covalent binding of nitroso dapsone to EBVtransformed B-cells ( Figure 7B) and the activation of clones with soluble nitroso dapsone.
In contrast, glutathione did not alter the activation of clones with nitroso dapsone-modified peptides, where the nitroso moiety is already bound covalently to the cysteine residue in the peptide sequence ( Figure 7C).

Activation of CD8+ T-cell clones with nitroso dapsone-modified peptides is HLA-B*13:01restricted
In in vitro culture conditions, soluble nitroso dapsone interacts with multiple HLA proteins to activate CD4+ and CD8+ clones from hypersensitive patients (14). The optimized culture conditions with extensive covalent modification of cellular protein potentially overrides the exquisite HLA restriction observed in patients. Thus, a stepwise approach was used to explore the restriction of the nitroso dapsone-modified peptide-specific T-cell response. First, with the exception of a small number of self-presenting clones, T-cells were not stimulated to proliferate with nitroso dapsone-modified peptides when antigen presenting cells (EBV-transformed B-cells) were excluded from the assays ( Figure 8A). Second, C1R-B13:01 antigen presenting cells, expressing HLA-B*13:01, but not the other HLA class I alleles expressed by the patients were used as antigen presenting cells in the place of autologous EBV-transformed B-cells. Clones were activated and secreted IFN-γ when cultured with either nitroso dapsone-modified peptides or soluble nitroso dapsone and C1R-B*13:01 cells ( Figure 8B). Third, IFN-γ secretion above control levels was not detected when the experiment was repeated with nitroso dapsone-modified Pep1 or Pep3 and C1R-parental cells ( Figure 9A). Finally, pre-treatment of C1R-B*13:01 antigen presenting cells with an anti-HLA class I blocking antibody inhibited peptide-induced IFN-γ secretion, whereas an anti-HLA class II blocking antibody had no effect ( Figure 9B).

Discussion
Delayed-typed drug hypersensitivity reactions are a serious form of adverse event and represent a challenge to healthcare professionals attempting to delineate patient susceptibility. Drug-responsive T-cells are believed to be the primary effector cells involved in the iatrogenic disease, with drug (metabolite) protein or peptide binding believed to be the molecular initiating event. This interaction may involve the drug molecule binding covalently to cellular or serum proteins, as is the case for β-lactam antibiotics such as flucloxacillin (18)(19)(20). The resultant adducts are thought to be processed by antigen presenting cells into peptide fragments that associate with HLA proteins for presentation to T-cells. At the opposite end of the spectrum, drugs such as carbamazepine form labile binding interactions with HLA proteins or peptides within the HLA antigen binding cleft to stimulate a similar effector T-cell response (21)(22)(23). It should be noted that although the pathways that lead to drug display by HLA proteins differ, the chemical composition and 3D arrangement of molecules at the immunological synapse may be similar with the only difference being the nature of the drug peptide binding interaction.
Different forms of drug hypersensitivity reaction are strongly associated with expression of specific HLA class I alleles (24)(25)(26). This suggests that a derivative of the drug may interact with exquisite selectivity with the protein encoded by the HLA allele to activate the T-cells that instigate the hypersensitivity reaction. Indeed, for the archetypal association between HLA-B*57:01 and abacavir hypersensitivity (27)(28)(29), the drug adheres deep within the peptide binding cleft of HLA-B*57:01, altering the structure and the peptides that are displayed by the HLA protein to CD8+ T-cells (30)(31)(32). A similar binding interaction is not observed with closely-related HLA proteins. Regrettably, the picture is not so clear for other forms of HLA class I allele-restricted forms of drug hypersensitivity reaction. Even with exemplars such as carbamazepine (HLA-B15:02 (33), HLA-A*31:01 (34)) and flucloxacillin (HLA-B*57:01 (35)) the parent drug, drug metabolites and/or peptide adducts interact with multiple HLA class I and class II proteins to stimulate CD4+ and CD8+ T-cells in hypersensitive patients (36)(37)(38)(39)(40)(41). In recent years we have focused on dapsone hypersensitivity to further define pathways of T-cell activation as (i) the metabolism and protein reactivity of dapsone is well defined (8,9,11), and (ii) dapsone hypersensitivity is strongly associated with HLA-B*13:01 expression (4). Dapsone and nitroso dapsone activate polyclonal CD4+ and CD8+ T-cells via different pathways, pharmacological HLA binding and hapten binding, respectively (13,14). A number of T-cells display dapsone and nitroso dapsone cross-reactivity; however, others are highly selective in that they are stimulated with one molecule and not the other. Thus, exposure of susceptible patients to parent drug and metabolite results in the development of divergent T-cell responses that act together produce the adverse event. HLA-B*13:01-restricted dapsone-and nitroso dapsone-responsive CD8+ T-cells are detectable in assays utilizing antigen presenting from donors expressing matching HLA-B alleles. Nitroso dapsone activates T-cells via two pathways. First, through direct covalent modification of peptides embedded within MHC expressed on the surface of antigen presenting cells; and, second, through formation of protein adducts that undergo antigen processing to generate peptides that associate with MHC before transport to the cell surface for presentation to T-cells. The same clone may be activated by both pathways with the surface peptide adduct presumably mimicking the adduct formed naturally through protein processing. No information is available regarding the nature of protein adducts that activate T-cells (including whether they are formed intra or extracellularly), the different uptake pathways involved in internalising adducts and the enzymes involved in breakdown of the adducts. For this reason, we have designed and synthesized nitroso dapsone-modified HLA binding peptides to study the HLA-B*13:01restricted T-cell response. Three nitroso dapsone-modified peptides were synthesized in high purity. Each peptide contained 2 HLA-B*13:01 anchoring motifs, an alanine backbone (previous studies show that non-anchoring amino acids for the most part do not define the specificity of the T-cell response (42,43)) and a nucleophilic cysteine residue for modification by nitroso dapsone. An Fmoc protecting group, which was removed before purification, was used to prevent nitroso dapsone N-terminal binding. Reasonable yields of nitroso dapsone-modified Pep1 and Pep3 were obtained after HPLC purification; Pep2 was generated in a lower quantity and as such only used in limited T-cell cross-reactivity studies. Pep1 contains a cysteine residue in the 4 position, which is an important trinitrophenol hapten binding site for the generation of immunodominant CD8+ peptide epitopes (44). In contrast, trinitrophenol modification at more distal positions (e.g., position 7 in Pep3) generates qualitatively different determinants that tend to activate a lower frequency of T-cells (44). Modelling revealed that the predicted conformation of dapsone-modified peptides contained the bulky drug aromatic groups pointing out the binding groove to ensue favourable interaction of P2 and P9 anchor residues with HLA-B*13:01.
Two lymphocyte transformation test positive (dapsone and nitroso dapsone) hypersensitive patients (described in (14)) expressing HLA-B*13:01 were used to generate nitroso dapsone-modified peptide responsive T-cell clones. Almost 50% of clones generated from 14-day nitroso dapsone-modified peptide PBMC cultures were stimulated to proliferate in the presence of either modified Pep1 or Pep3. Clones expressed a CD8+ phenotype and were stimulated with the modified peptides in a dose-dependent manner. Clones displayed 100% cross-reactivity between positional derivatives and with soluble nitroso dapsone. Nitroso dapsone extensively modified the surface of antigen presenting cells, which likely includes binding to cysteine-containing peptides already displayed by HLA class I on the cell surface; hence, the observed cross-reactivity was expected. In contrast to our cross-reactivity data with nitroso dapsone-modified peptides, cross-reactivity between HLA class II binding βlactam-modified peptide positional derivatives was not observed (16,45). However, a system utilized by Honda et al (46), where CD8+ T-cell receptor α and β chains were alternately fixed prior to assessment of trinitrophenol-modified peptide positional derivative T-cell responses explains how a single T-cell receptor is capable of recognizing and responding to hapten structures in different positions. They demonstrated that hapten addition to HLA class I binding peptides (at positions 4 and 6) can cause substantial adjustments to the CD8+ T-cell receptor structure Specifically, the β chain could adjust to interact with the hapten structure irrespective of whether it was at position 4 or 6.
The possibility that the T-cell activation with nitroso dapsone-modified peptides may be due to residual soluble nitroso dapsone or degradation of the adduct in culture and liberation of dapsone was excluded through (i) demonstrating that the clones were not activated with the parent compound, and (ii) neutralizing soluble nitroso dapsone-specific, but not nitroso dapsone-modified peptide-specific, T-cell responses with glutathione. Glutathione contains a reactive cysteine group and when in excess binds to nitroso dapsone, preventing formation of protein adducts.
To explore the importance of HLA proteins in T-cell activation, antigen presenting cells were firstly excluded from T-cell proliferation assays. The vast majority of clones were not activated with the nitroso dapsone-modified peptides in the absence of antigen presenting cells. Next, the HLA-A, B negative mutant C1R cell line was transduced with HLA-B*13:01 and used as antigen presenting cells in the place of autologous EBV-transformed B-cells. Nitroso dapsone-modified Pep1 and Pep3, and soluble nitroso dapsone stimulated the clones to secrete IFN-γ in the presence of C1R-B*13:01 cells and the response was inhibited with an HLA class I blocking antibody. Similar activation of the T-cell clones was not observed when using the C1R parental cell line as antigen presenting cells.
Collectively, our study highlights the importance of drug metabolism, drug hapten binding and most importantly the formation of drug metabolite-modified HLA-B*13:01 binding peptides in the activation of CD8+ T-cells from dapsone hypersensitive patients. The availability of T-cell stimulatory nitroso dapsone-modified HLA-B*13:01 binding peptides and C1R-B*13:01 cells offer the opportunity to study the structural elements of the drug hapten HLA peptide binding interaction.    A panel of up to 30 nitroso dapsone-modified peptide-responsive CD8+ clones were used to explore cross-reactivity with unmodified peptide, soluble nitroso dapsone and modified peptides with cysteine located in different positions in the peptide sequence. Clones (  (A) Nitroso dapsone-modified peptide-responsive CD8+ clones (5 × 10 4 cells/well; 200 μl) were incubated with irradiated autologous antigen presenting cells (EBV-transformed B-cells; 1 × 10 4 cells/well) and nitroso dapsone-modified Pep1, Pep2 or Pep3 for 48 hrs. IFN-γ release was measured by ELIspot. To demonstrate that the T-cell clones secreted a profound T-cell response, secreted cytokines were measured from 2 clones that were stimulated to proliferate and 2 that did not (B) using ELIspot (C) and a cytokine bead array (D and E). The clones that proliferated and secreted IFN-γ measured by ELIspot, were   the presence or absence of anti-HLA class I and II blocking antibodies for 48 hrs. IFN-γ release was measured by ELIspot.